published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Despite recognition of aldehyde dehydrogenase (Aldh) as a surrogate marker in isolating primitive hematopoietic stem and progenitor cells (HSPC) [1], its role in HSPC regulation, particularly during embryonic development, remains unclear. In this study, we examined the role of Aldh during embryonic hematopoiesis in zebrafish, which has emerged as a model for hematopoietic studies. [2] Wild--?type and transgenic [Tg(gata1:gfp),Tg(fli1:gfp)] zebrafish embryos were microinjected with anti--?sense morpholinos (MO) at 1--?cell to 4--?cell stage and evaluated by morphology, flow cytometry, in situ hybridization (ISH) and Q-RT-PCR. In addition, human CD34+ cells, which were enriched with hematopoietic stem cells (HSC), were isolated from umbilical cord blood samples for analysis of ALDH16A1 expression. It was subsequently compared with CD34- cells which were devoid of HSC activity. When aldh16a1 was knocked down by anti-sense morpholino (the embryos were referred herewith aldh16a1MO embryos), gene expression associated with erythropoiesis was significantly reduced at 18hpf .(gata1:0.70±0.03fold; p=0.002) (α-embryonic hemoglobin: 0.48±0.04fold; p=0.003) (β-embryonic hemoglobin: 0.56±0.03fold; p=0.001). Angiogenesis was also perturbed at 48 and 72hpf. Furthermore, human ALDH16A1 was significantly upregulated (4.79±1.00fold; p=0.00006) in CD34+ (enriched with HSC) as compared to CD34- (devoid of HSC) populations in umbilical cord blood. Aldh16a1 is important for the maintenance of primitive hematopoiesis at early (18hpf) and angiogenesis at later (48,72 hpf) embryonic stages. As angiogenesis plays an important role in pathophysiology of malignancies, novel therapy against ALDH16A1 might be exploited in therapeutic intervention in cancer treatment. Moreover, a specific role of zebrafish aldh16a1 in primitive erythropoiesis and a higher level of ALDH16A1 expression in human HSC-enriched cells suggested a conserved mechanism whereby ALDH regulates hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Bibliography: leaves 150-178. / xxii, 178 leaves, [17] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the potential of the zebrafish Danio rerio to provide a convenient, fecund and cost efficient piscine model for biotechnology. The technical and biological limitations of the model are identified and ways of overcoming or circumventing these limitations explored in three areas of biotechnology: transgenesis; partial genome manipulation; and, entire genome manipulatiion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics & Gynaecology, 1996?

Bibliography: leaves 150-178. / xxii, 178 leaves, [17] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the potential of the zebrafish Danio rerio to provide a convenient, fecund and cost efficient piscine model for biotechnology. The technical and biological limitations of the model are identified and ways of overcoming or circumventing these limitations explored in three areas of biotechnology: transgenesis; partial genome manipulation; and, entire genome manipulatiion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics & Gynaecology, 1996?

No description available.

The development of spinal circuits underlying motor behaviors was examined in zebrafish. Zebrafish embryos showed three sequential, stereotyped behaviors: a transient period of spontaneous coiling contractions, followed by touch-evoked rapid coils, and swimming. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. / The first (spontaneous) behavior was chosen for further analysis in order to characterize the underlying circuit. In vivo patch clamp recordings were obtained from identified spinal neurons. These neurons showed periodic depolarizations that triggered rhythmic bursts of action potentials with a frequency and duration that were consistent with those of the spontaneous contractions. As with the behavior, transecting the spinal cord at the hindbrain border did not affect the rhythmic activity patterns of the neurons. Surprisingly the contractions and the periodic depolarizations were insensitive to both general and specific blockade of synaptic transmission. The periodic depolarizations were suppressed by heptanol and by intracellular acidification treatments that are known to uncouple gap junctions, indicating that electrotonic synapses could underlie network synchronization during the earliest motor behavior. / Paired recordings were obtained from identified spinal neurons. These showed that active ipsilateral neurons were electrically coupled in a simple network consisting initially of motoneurons and only three types of interneurons. Therefore, this early spinal circuit consists of rhythmically active and electrically coupled neurons. Furthermore, this circuit is also initially independent of the main neurotransmitter systems, sensory inputs, and descending hindbrain projections. The descending projections are required later in development for the onset of touch responses and swimming.

Many studies addressing the toxicity of silver nanomaterials have found that smaller sized silver nanoparticles are usually more toxic to organisms and in cell culture than particles of larger sizes yet it is not entirely clear why. We investigated the size dependent toxicity of silver nanoparticles by measuring the response of embryonic zebrafish (Danio rerio) following exposure to a library of thirteen distinct silver nanoparticle size distributions with mean diameters between 8.9 nm and 112.6 nm. Data analysis using dose���response modeling revealed that silver nanoparticles (AgNP) induced embryo toxicity that is dependent on the total surface area and not on the mass or particle number in solution. Included in this study is a comparison between embryo toxicity induced by silver nitrate (AgNO���) and AgNPs for cardiovascular endpoints, as well as an investigation into the influence of the chorion on AgNP toxicity. This study demonstrates the importance of using alternative dose metrics in nanotoxicology, and highlights the value of using the embryonic zebrafish to explore nanomaterial structure activity relationships. / Graduation date: 2013

Vitamin E was first described in 1922 as an unknown factor required for impregnated rats to carry their offspring to term. In fact, when vitamin E was chemically characterized it was given the name "tocopherol" derived from the Greek: tokos = childbirth; phero = to bear; and –ol, indicating an alcohol. Vitamin E is linked to animal health and wellness, maternal fertility and a human neurodegenerative condition, ataxia with vitamin E deficiency However, embryonic vitamin E requirements during development remained unknown. We hypothesized that vitamin E is critical, not only for the mother, but specifically by the embryo for proper development. To separate the embryonic and maternal requirements, we employed an innovative model for the study of vitamin E: the zebrafish. We began by formulating and testing the first fully defined diet sufficient for zebrafish health. We then removed vitamin E from the formula to create our E deficient (E-) diet, which, when fed to adult zebrafish (for >3 months), resulted in E- adults that produced viable, E- gametes. Deficient embryos initially developed normally; however, by 48 hours post fertilization (hpf), E- embryos developed severe malformations leading to significant mortality. Thus, we demonstrated for the first time an embryonic vitamin E requirement. We provided further insight into the embryonic vitamin E requirement by analyzing the transcriptional changes occurring prior to the observed malformations. The transcriptome revealed a putative mechanism of action for vitamin E in development, in which vitamin E deficiency leads to the dysregulation of key metabolic co-activators. Finally, to understand the trafficking of vitamin E, we identified the zebrafish α-tocopherol transfer protein (TTP). We demonstrated that the zebrafish TTP is homologous to its human counterpart, and its expression is both spatially and temporally regulated during embryonic development. Knocking down the expression of TTP, using morpholinos injected at the one-cell stage, resulted in early and severe malformations in the developing head and tail. Consequently we revealed a definitive role for TTP during development. Taken together the work described here presents a new direction for future research into the role of vitamin E and TTP in post-implantation development. / Graduation date: 2012